The present invention relates to an in vitro cell culture including cartilage, as well as various uses thereof, including screening for compounds which can modify cell/cell or cartilage/cell interactions.
Restoration of epithelial tissue after tissue injury is a complex process, which includes several critical events, including deposition of extracellular matrix (xe2x80x9cECMxe2x80x9d), tissue remodeling, and angiogenesis. These events are coordinated with epithelial cell migration and proliferation to restore the epithelial and/or mucosal barrier (i.e., in epithelial tissues such as tracheal epithelium which secrete mucous). The coordination of these events is believed to involve the interaction between different classes of cells as well as between cells and their extracellular matrix.
Failure of reepithelialization after injury has been observed in the cornea (Fini et al., xe2x80x9cExpression of Collagenolytic/Gelatinolytic Metalloproteinases by Normal Cornea,xe2x80x9d Invest. Opthalmol. Vis. Sci. 31:1779-1788 (1990)) and in chronic wounds (Stacey et al., xe2x80x9cTissue and Urokinase Plasminogen Activators in the Environs of Venous and Ischemic Leg Ulcers,xe2x80x9d Br. J. Surg. 80:595-599 (1993); Wysocki et al., xe2x80x9cWound Fluid from Chronic Leg Ulcers Contains Elevated Levels of Metalloproteinases MMP-2 and MMP-9,xe2x80x9d J. Invest. Dermatol. 101:64-68 (1993); Madlener et al., xe2x80x9cMatrix Metalloproteinases (MMPs) and their Physiological Inhibitors (THAPs) are Differentially Expressed During Excisional Skin Wound Repair,xe2x80x9d Exp. Cell Res. 242:201-210 (1998); Di Colandrea et al., xe2x80x9cEpidermal Expression of Collagenase Delays Wound-healing in Transgenic Mice,xe2x80x9d J. Invest. Dermatol. 11:1029-1033 (1998)). Proteinases that destroy the basement membrane over which epithelial cells migrate have been implicated as mediators in impaired capacity to reepithelialize.
Tissue remodeling during wound healing is critical for repair as cellular migration over an appropriate ECM requires controlled and tightly regulated proteolytic degradation of the ECM, with consequent activation or release of matrix-bound growth factors (Clark, xe2x80x9cBasics of Cutaneous Wound Repair,xe2x80x9d J. Dermatol. Surg. Oncol. 19:693-706 (1993); Salo et al., xe2x80x9cExpression of Matrix Metalloproteinase-2 and -9 During Early Human Wound Healing,xe2x80x9d Lab. Invest. 70:176-182 (1994); Vaalamo et al., xe2x80x9cPatterns of Matrix Metalloproteinase and TIMP-1 Expression in Chronic and Normally Healing Human Cutaneous Wounds,xe2x80x9d Br. J. Dermatol. 135:52-59 (1996); Moses et al., xe2x80x9cTemporal Study of the Activity of Matrix Metalloproteinases and Their Endogenous Inhibitors During Wound Healing,xe2x80x9d J. Cell. Biochem. 60:379-386 (1996); Martin xe2x80x9cWound Healingxe2x80x94Aiming for Perfect Skin Regeneration,xe2x80x9d Science 276:75-81 (1997); Arumagam et al., xe2x80x9cTemporal Activity of Plasminogen Activators and Matrix Metalloproteinases During Cutaneous Wound Repair,xe2x80x9d Surgery 125:5887-593 (1999)).
As with the above-described tissues, reepithelialization of injured tracheal tissues is often incomplete. The ability of respiratory epithelial cells (xe2x80x9cRECsxe2x80x9d) to migrate and proliferate and restore denuded areas of the large conducting airway after injury is poor. Post-trauma restoration is pathologically manifested by the exuberant proliferation of granulation tissue and replacement of the normal respiratory epithelium with fibroblasts (Clark, xe2x80x9cThe Commonality of Cutaneous Wound Repair and Lung Injury,xe2x80x9d Chest. 99(Suppl.):57S-60S (1991); Grillo, xe2x80x9cTracheal Replacement,xe2x80x9d Ann. Thorac. Surg. 49:864-865 (1990)). This often leads to scar formation, airway stenosis, and eventual physiologic compromise of the host respiratory tract.
There is currently no effective way to study events of reepithelialization after injury, particularly with respect to the intraluminal events surrounding tracheal repair. Present approaches to tracheal repair include resection and reanastomosing the injured airway, replacement of the damaged portion by synthetic material, and use of autologous tissue for reconstruction of the tracheal defect (Letang et al., xe2x80x9cExperimental Reconstruction of the Canine Trachea with a Free Revascularized Small Bowel Graft,xe2x80x9d Ann. Thorac. Surg. 49:955-958 (1990); Mulliken et al., Abstract, xe2x80x9cThe Limits of Tracheal Resection with Primary Anastomosis: Further Anatomical Studies in Man,xe2x80x9d J. Thorac. Cardiovasc. Surg. 55:418 (1968); Neville et al., xe2x80x9cProsthetic Reconstruction of the Trachea and Carina,xe2x80x9d J. Thorac. Cardiovasc. Surg. 72:525-536 (1976)). Recently, tissue engineering approaches have been taken, including forming an in vivo tracheal cartilaginous scaffolding by injecting dissociated chondrocytes into a preformed synthetic construct (Hirano et al., xe2x80x9cHydroxylapatite for Laryngotracheal Framework Construction. Ann. Otol. Rhinol. Laryngol. 98:713-717 (1989); Okumura et al., xe2x80x9cExperimental Study of a New Tracheal Prosthesis Made from Collagen Grafted Mesh,xe2x80x9d Trans. Am. Soc. Artif. Organs. 37:M317-M319 (1991); Langer et al., xe2x80x9cTissue Engineering,xe2x80x9d Science 260:920-926 (1993)). Such devices were of limited success owing to lack of reepithelialization. In the case of synthetic replacement, migration of the prosthesis can occur and may result in chronic ulceration, and even fatal hemorrhage (Grillo, xe2x80x9cTracheal Replacement,xe2x80x9d Ann. Thorac. Surg. 49:864-865 (1990)).
A frequent problem seen in tracheal repair with synthetic or autologous materials is the failure of luminal surface reepithelialization. Failure of reepithelialization to reestablish luminal integrity is an important reason why no acceptable surgical procedure exists for the repair of extended segments of trachea compromised by inhalation injury, congenital anomalies, or neoplastic disease.
Why the rate of reepithelialization in the large conducting airway is different from that seen within other epithelial-lined or -covered surfaces is unclear. The phenomenon of xe2x80x9cslowedxe2x80x9d reepithelialization is seen after both ablative surgical reconstruction and denudation injury, where the epithelium and basement membrane are removed with an intact cartilaginous superstructure (e.g., inhalation injury).
One of the difficulties in understanding the relationship between epithelium and its underlying substructure (cartilage and submucosa) is the inaccessibility of the tissue for direct observation. It would be desirable, therefore, to provide an in vitro cell culture which includes a developed substructure or cartilaginous layer which can be used to study epithelial cell development.
The present invention is directed to overcoming these and other deficiencies in the art.
One aspect of the present invention relates to an in vitro cell culture device which includes a vessel including an inner surface, a layer of cartilage disposed on at least a portion of the inner surface, the layer of cartilage including a plurality of chondrocytes in an extracellular matrix, and a growth medium in the vessel, the layer of cartilage being bathed in the growth medium.
A further aspect of the present invention relates to a composite cell culture which includes a first layer including chondrocytes in an extracellular matrix, a second layer disposed on the first layer and including type I collagen, and a third layer disposed on the second layer and including cells at least partially covering the second layer.
Another aspect relates to a method for preparing an in vitro composite cell culture. This method is carried out by providing an in vitro cartilage layer that includes chondrocytes in an extracellular matrix, disposing a type I collagen layer on the cartilage layer, and contacting the type I collage layer with epithelial cells under conditions effective for the epithelial cells to multiply and at least partially cover the layer of type I collagen.
Still another aspect of the present invention relates to a method of screening putative therapeutic agents for activity in promoting re-epithelialization of cartilaginous tissues. According to one approach, the method is carried out by introducing a putative therapeutic agent into a composite cell culture of the present invention and then assessing epithelial cell growth on the composite cell culture, wherein increased surface area coverage of a plurality of distinct plaques of epithelial cells indicates that the putative therapeutic agent has activity in promoting re-epithelialization of cartilaginous tissues. Alternatively, this method is carried out by providing an in vitro cell culture device of the present invention, introducing a layer of type I collagen onto the layer of cartilage, introducing epithelial cells onto the layer of type I collagen to form a composite cell culture, introducing a putative therapeutic agent into the composite cell culture, and assessing epithelial cell growth on the composite cell culture, wherein growth and migration of epithelial cells beyond distinct plaques thereof indicates that the putative therapeutic agent has activity in promoting re-epithelialization of cartilaginous tissues.
Yet another aspect of the present invention related to a method of screening putative therapeutic agents for activity in inhibiting a growth factor or proteinase which prevents re-epithelialization of cartilaginous tissues. According to one approach, this method is carried out by introducing a putative therapeutic agent into a composite cell culture of the present invention and assessing epithelial cell growth on the composite cell culture, wherein increased surface area coverage of a plurality of distinct plaques of epithelial cells indicates that the putative therapeutic agent has activity in inhibiting a growth factor or proteinase which prevents re-epithelialization. Alternatively, this method is carried out by providing an in vitro cell culture device of the present invention, introducing a layer of type I collagen onto the layer of cartilage, introducing epithelial cells onto the layer of type I collagen to form a composite cell culture, introducing a putative therapeutic agent into the composite cell culture, and assessing epithelial cell growth on the composite cell culture, wherein growth and migration of epithelial cells beyond distinct plaques thereof indicates that the putative therapeutic agent has activity in inhibiting a growth factor or proteinase which prevents re-epithelialization.
The in vitro cell culture device of the present invention enables the growth of cells on an in vitro cartilage substructure, which enables the study of cell-cell interactions between chondrocytes and other cell types introduced onto the cell culture device, as well as cell-matrix interactions between cartilage and other cell types introduced onto the cell culture device. When collagen inserts are placed onto the cell culture device and isolated cells or tissues are introduced onto the collagen inserts, the resulting composite cell culture can similarly be used. In addition, the in vitro cell culture device and composite cell culture can be used to screen various therapeutic agents for their ability to modify such cell-cell or cell-matrix interactions, both on a cellular level as well as on a molecular level. The in vitro cell culture device and composite cell culture will facilitate the development of systems in which graft tissues can be raised in vitro for subsequent grafting onto a patient, preferably using the patient""s own cells so as to avoid any undesirable immune reactions.